WO1994021660A1 - Recepteur alpha1c-adrenergique humain clone - Google Patents

Recepteur alpha1c-adrenergique humain clone Download PDF

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WO1994021660A1
WO1994021660A1 PCT/US1994/002609 US9402609W WO9421660A1 WO 1994021660 A1 WO1994021660 A1 WO 1994021660A1 US 9402609 W US9402609 W US 9402609W WO 9421660 A1 WO9421660 A1 WO 9421660A1
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Prior art keywords
human
receptor
leu
val
ser
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PCT/US1994/002609
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English (en)
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Marvin L. Bayne
Bradley V. Clineschmidt
Catherine D. Strader
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Merck & Co., Inc.
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Priority to JP6521115A priority Critical patent/JPH08508163A/ja
Priority to AU64453/94A priority patent/AU685789B2/en
Priority to EP94912209A priority patent/EP0689547A4/fr
Publication of WO1994021660A1 publication Critical patent/WO1994021660A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to a method for defining the potency and selectivity of compounds for use as human alphalC adrenergic receptor antagonists using cloned human alpha 1 receptors.
  • the invention also relates to the cloned receptors themselves, to compounds identified according to the method of this invention, and to methods of use of such compounds, alone or in combination with other agents.
  • Particularly preferred are combinations of compounds identified according to this invention and testosterone 5-alpha reductase inhibitors, to alleviate pathologic conditions, particularly benign prostatic hyperplasia (also known as benign prostatic hypertrophy, BPH).
  • Human adrenergic receptors are integral membrane proteins which have been classified into two broad classes, the alpha and the beta adrenergic receptors. Both types mediate the action of the peripheral sympathetic nervous system upon binding of catecholamines, norepinephrine and epinephrine.
  • Norepinephrine is produced by adrenergic nerve endings, while epinephrine is produced by the adrenal medulla.
  • the binding affinity of adrenergic receptors for these compounds forms one basis of the classification: alpha receptors bind norepinephrine more strongly than epinephrine and much more strongly than the synthetic compound isoproterenol.
  • the binding affinity of these hormones is reversed for the beta receptors.
  • the functional responses such as smooth muscle contraction, induced by alpha receptor activation are opposed to responses induced by beta receptor binding.
  • alpha and beta receptors were further subdivided into oq, cc2, Bl, and B2 subtypes. Functional differences between c and ⁇ 2 receptors have been recognized, and compounds which exhibit selective binding between these two subtypes have been developed.
  • WO 92/0073 the selective ability of the R(+) enantiomer of terazosin to selectively bind to adrenergic receptors of the alpha 1 subtype was reported.
  • the ⁇ i/ ⁇ 2 selectivity of this compound was disclosed as being significant because agonist stimulation of the OA ⁇ receptors was said to inhibit secretion of epinephrine and norepinephrine, while antagonism of the o 2 receptor was said to increase secretion of these hormones.
  • non- selective alpha-adrenergic blockers such as phenoxybenzamine and phentolamine, is limited by their ⁇ 2 adrenergic receptor mediated induction of increased plasma catecholamine concentration and the attendant physiological sequelae (increased heart rate and smooth muscle contraction).
  • Benign prostatic hypertrophy is an illness typically affecting men over fifty years of age, increasing in severity with increasing age.
  • the symptoms of the condition include, but are not limted to, increased difficulty in urination and sexual dysfunction. These symptoms are induced by enlargement, or hypertrophy, of the prostate gland. As the prostate increases in size, it impinges on free-flow of fluids through the male urethra. Concommitantly, the increased noradrenergic innervation of the enlarged prostate leads to an increased adrenergic tone of the bladder neck and urethra, further restricting the flow of urine through the urethra.
  • the mechanism of prostatic hypertrophy is well understood.
  • the male hormone, 5 ⁇ -dihydrotestosterone has been identified as the principal culprit.
  • the continual production of 5 ⁇ - dihydrotestosterone by the male testes induces incremental growth of the prostate gland throughout the life of the male. Beyond the age of about fifty years, in many men, this enlarged gland begins to obstruct the urethra with the pathologic symptoms noted above.
  • one solution is to identify pharmaceutically active compounds which complement slower-acting therapeutics by providing acute relief.
  • Agents which induce relaxation of the urethral smooth muscle, by binding to alpha- 1 adrenergic receptors, thus reducing the increased adrenergic tone due to the disease, would be good candidates for this activity.
  • one such agent is alf ⁇ zosin, which is reported in EP 0 204597 to induce urination in cases of prostatic hypertrophy.
  • the selective ability of the R(+) enantiomer of terazosin to bind to adrenergic receptors of the ⁇ i subtype was reported.
  • identification of active compounds is through use of animal tissues known to be enriched in adrenergic receptors.
  • rat tissues have been used to screen for potential adrenergic receptor antagonists.
  • compounds which appear active in animal tissue may not be active or sufficiently selective in humans. This results in substantial wastage of time and effort, particularly where high volume compound screening programs are employed.
  • compounds, which might be highly effective in humans would be missed because of their absence of appreciable affinity for the heterologous animal receptors.
  • even single amino acid changes between the sequence of biologically active proteins in one species may give rise to substantial pharmacological differences.
  • the instant inventors have solved these problems by cloning a novel human adrenergic receptor of the ⁇ ic subtype. Their efforts have led to the development of a novel screening assay which enables them to identify compounds which specifically interact with the human ⁇ lC adrenergic receptor. Marshall et al (Br. J. Pfiarm.. 107:327 (1992)) speculated that compounds which specifically interact with the ⁇ lC adrenergic receptor may be responsible for contraction of the human prostate.
  • the instant invention provides a method for identifying compounds which bind the human ⁇ ic receptor. In addition, if the compounds are further tested for binding to other human alpha 1 receptor subtypes, as well as counterscreened against other types of receptors, the specificity of the compounds for the human ⁇ ic adrenergic receptor may be defined.
  • Compounds identified according to this invention may be used to reduce the acute symptoms of BPH. New agents identified in this manner, or already known agents showing activity in this assay, may now be employed in a novel way to help BPH sufferers contend with the acute symptoms of the syndrome. Thus, this invention is useful to identify compounds which may be used alone or in conjunction with a more long-term anti-BPH therapeutics, such as PROSCAR®. Other uses for the invention include identification of compounds which induce highly tissue-specific, localized ⁇ ic adrenergic receptor blockade. Effects of this blockade include reduction of intra-ocular pressure, control of cardiac arrhythmias, and possibly a host of alpha- IC receptor mediated central nervous system events.
  • the cloned ⁇ ic receptor can be used for screening of tissue specific expression of ⁇ ic adrenergic receptors. Effects such as these, induced by or available to analysis with the ⁇ ic adrenergic receptor also form part of this invention.
  • the human adrenergic receptor of the alphalC subtype is cloned and used in an in vitro assay to screen for compounds that bind to the receptor, including compounds which specifically inhibit the activity of the receptor.
  • the invention includes the assay, the cloned receptor used in the assay (cDNA), an isolated human alphalC adrenergic receptor, cells expressing the cloned receptor, and compounds identified through the use of this novel, cloned receptor, which selectively bind to the human alphalC adrenergic receptor, including specific antagonists of the receptor.
  • One embodiment of this invention is a method of treating benign prostatic hypeplasia (BPH) employing compounds having an affinity for the human alpha IC receptor that is at least 12 fold greater than for either the human alpha 1A or the human alpha IB receptors.
  • Fig. 1 Sequence of cDNA obtained by PCR of human heart mRNA, SEQ. ID:4:.
  • Fig. 2 Comparison of the open reading frame obtained from human heart, SEQ ID:5:, and the bovine alpha-lC adrenergic receptor sequence, SEQ. LD:6:.
  • Fig. 3 Sequence of cDNA obtained by screening a human hippocampus cDNA library using the heart mRNA derived sequence from figure 1, SEQ. LD:7:
  • Fig. 4 Sequence of 3' coding region of human alpha- IC gene, obtained by PCR amplification of a human genomic DNA library with oligonucleotides, SEQ. ID:10:.
  • Fig. 5 Sequence of the ligated portions of human alpha- IC DNA shown in figures 3 and 4, SEQ. ID: 11:.
  • Fig. 6 The amino acid sequence of the human alpha- IC adrenergic receptor, SEQ ID: 12:.
  • Fig. 7 The alignment of the nucleotide and amino acid sequence of the human alpha- IC adrenergic receptor, showing the 5'-untranslated region, SEQ. ID:11: and SEQ. ID: 12:.
  • Fig. 8 Expression of the human alpha- IC adrenergic receptor in COS cells: Binding data using membranes from cells transfected with the expression vector alone and the expression vector containing the human alpha- IC adrenergic receptor coding sequences.
  • Fig. 9 Binding curves of compounds using membranes from COS cells transfected with the human alpha- IC adrenergic receptor containing expression vector.
  • Fig. 10 Nucleotide sequence of the human alphal A receptor, SEQ. ID:13:
  • Fig. 11 Amino acid sequence of the human alphal A adrenergic receptor, SEQ. ID: 14:
  • Fig. 12 Partial sequence of the human alpha IB adrenergic receptor, SEQ. ID:17:
  • Fig. 13 Partial sequence of the human alphal B adrenergic receptor, SEQ. ID:20:
  • Fig. 14 Partial sequence of the human alphalB adrenergic receptor, SEQ. ID:23:
  • Fig. 15 Composite human/rat alphalB adrenoreceptror, SEQ. ID:24:
  • Fig. 16 Amino acid sequence of the composite human/rat alphalB adrenergic receptor, SEQ. LD:25:
  • Fig. 17 Binding curves of compounds using membranes from COS cells transfected with the human alphalA, IB, and IC adrenergic receptor expression vectors.
  • Fig. 18 Sequence of truncated human alphalC adrenergic receptor, SEQ. LD:26:.
  • Fig. 19 Nucleotide sequence of the human ⁇ lC adrenergic receptor having a Pstl site, SEQ.LD:27:.
  • Fig. 20 Amino acid sequence of the human ⁇ lC adrenergic receptor encoded by the Pstl site encoding allele, SEQ.ID:28:.
  • Fig. 21 Alignment of the nucleotide and amino acid sequences of figures 19 and 20, SEQ.LD:27: and SEQ.ID:28:.
  • FIG. 22 Nucleotide sequence of the human ⁇ iA adrenergic receptor, Seq.ID:29:.
  • Fig. 23 Amino acid sequence of the human ⁇ i A adrenergic receptor, SEQ.ID:30:.
  • Fig. 24 Alignment of the nucleotide and amino acid sequences of figures 22 and 23, SEQ.LD:29: and SEQ.ID:30:.
  • the human alpha adrenergic receptor of the 1-C subtype was identified, cloned and expressed by the instant inventors.
  • a partial coding region for this receptor was generated by reverse transcriptase- polymerase chain reaction technology, RT-PCR. Accordingly, degenerate oligonucleotides encoding amino acids conserved in the fifth and sixth transmembrane domains of all three ⁇ l receptor subtypes (A, B, C) were used to prime RT-PCR reactions using human heart mRNA as template. The predicted sized products were cloned and sequenced. Translation of the amplified cDNA yielded an open reading frame encoding a protein 95% homologous to the bovine ⁇ lC receptor (Fig.2, SEQ.
  • SEQ. LD:6 This partial sequence was used to obtain a larger cDNA clone from a human hippocampus library (Fig. 3, SEQ. LD:6:). The remaining coding region was obtained by PCR amplification of human genomic DNA using primers based on the cDNA sequence and the last six amino acids of bovine ⁇ lC receptor (Fig. 4, SEQ.LD:10:). The complete receptor was then assembled using the partial sequences shown in Fig. 3, SEQ._D:6: and Fig. 4, SEQ. ID:10:, to generate the sequence shown in Fig. 5, SEQ. ID: 11:. The translation of this sequence is shown in Fig. 6, SEQ. ID: 12:, and the alignment of the nucleotide and amino acid sequences, and the 5'- untranslated sequences, is shown in Fig. 7, SEQ. LD:11: and SEQ. ID:12:.
  • the cloned human ⁇ lC receptor when expressed in mammalian cell lines (see Fig. 8), is used to discover ligands that bind to the receptor and alter its function.
  • the cloned ⁇ lC receptor enables quanititation of mRNA levels in human tissues, including the aorta and prostate, by RNase protection assays.
  • a complete coding sequence of the receptor is provided.
  • truncation at the 3' end of the sequence does not affect the functioning of the receptor.
  • SEQ. ID: 11 a sequence, truncated at the 3' end, SEQ. ED:26: is disclosed, which consists entirely of human alphalC sequence.
  • the specificty of binding of compounds showing affinity for the ⁇ lC receptor is shown by comparing affinity to membranes obtained from COS cells tranfected with the cloned ⁇ lC receptor and membranes from tissues known to express other types of alpha or beta adrenergic receptors.
  • the cloned human ⁇ iA and a hybrid human/rat ⁇ iB could be used for this pmpose, along with the human ⁇ lC receptor expressed in COS cells .
  • Expression of the cloned human ⁇ iA, ⁇ iB, and ⁇ lC receptors and comparison of their binding properties with known selective antagonists provides a rational way for selection of compounds and discovery of new compounds with predictable pharmacological activities.
  • the human receptor is cloned and expressed in a cell such as COS cells or CHO cells, the receptor is free of other human proteins.
  • the membranes from cells expressing different human alpha adrenergic receptor subtypes are then isolated according to methods well known in the art for membrane associated receptor binding assays. For example, the method of Schwinn, et al., (J. Biol. Chem.. 265:8183- 8189, 1990) may be used.
  • a compound of interest is used to compete with the binding of a known, quantifiable alpha receptor ligand.
  • radiolabled prazosin, niguldipine, 5-methyl urapidil, terazosin, dozazosin, phenoxybenzamine, WB4101, benoxathian, HEAT (2-[ ⁇ -(4- hydroxy-3-iodophenyl)ethylaminomethyl]tetralone, or phentolamine may be used for this pu ⁇ ose (see, for example, Robert R. Ruffolo, Jr., q-Adrenoreceptors: Molecular Biology. Biochemistry and Pharmacology. (Progress in Basic and Clinical Pharmacology series, Karger, 1991), page 29).
  • 125I-HEAT may be preferred for this pu ⁇ ose.
  • the labeled compound is competed off the receptor. From these experiments, IC50 values for each test compound and receptor subtype is determined.
  • a method for identifying compounds specific for the human alphalC receptor comprising the following steps:
  • a Cloning the human alphalC adrenergic receptor; b. Splicing the the cloned alphalC adrenergic receptor into an expression vector to produce a construct such that the alphalC receptor is operably linked to transcription and translation signals sufficient to induce expression of said receptor upon introduction of said construct into a prokaryotic or eukaryotic cell; c. Introducing said construct into a prokaryotic or eukaryotic cell which does not express a human alphalC adrenergic receptor in the absence of said introduced construct; d. Incubating cells or membranes isolated from cells produced in step c.
  • a quantifiable compound known to bind to human alpha adrenergic receptors and subsequently adding test compounds at a range of concentrations so as to compete the quantifiable compound from the receptor, such that an IC50 for the test compound is obtained as the concentration of test compound at which 50% of the quantifiable compound becomes displaced from the receptor; e. Incubating cells or membranes of cells which naturally express or have an introduced, cloned human alpha adrenregic receptor of a subtype other than the human alphalC receptor under identical conditions to the incubation conducted in step d, and obtaining the IC50 of the test compound for the non-alphalC receptor; and f. Comparing the IC50 for the test compound for the alphalC receptor and for the alpha adrenergic receptor of a subtype other than the alphalC to identify compounds having a lower IC50 for the alphalC receptor.
  • the instant inventors have also discovered a different sequence than that reported by Bruno et al., rBBRC 179:1485- 1490 (1991)] for the human ⁇ iA adrenergic receptor.
  • the new sequence is more homologous to the rat ⁇ iA adrenergic receptor sequence.
  • Disclosed and claimed herein is the sequence for this new human ⁇ iA adrenergic receptor (see Example 12 and figures 22, 23, and 24, SEQ. 1D:29: and SEQ. 1D:30:).
  • Compounds identified according to the method of this invention as being selective human ⁇ lC adrenergic receptor antagonists may further be defined by counterscreening. This is accomplished, according to methods known in the art using other receptors responsible for mediating diverse biological functions. Compounds which are both selective amongst the various human alphal adrenergic receptor subtypes and which have low affinity for other receptors, such as the alpha2 adrenergic receptors, the ⁇ -adrenergic receptors, the muscarinic receptors, the serotonin receptors, and others are particularly preferred.
  • Compounds identified according to this patent disclosure may be used alone at appropriate dosages defined by routine testing in order to obtain optimal inhibition of the human ⁇ lC adrenergic receptor while minimizing any potential toxicity.
  • co- administration or sequential administration of other agents which alleviate the effects of BPH is desirable.
  • this includes administration of compounds identified according to this disclosure and a human testosterone 5- ⁇ reductase inhibitor.
  • Many such compounds are now well known in the art and include such compounds as PROSCAR®, (also known as finasteride, a 4-Aza-steroid; see US Patents 4,377,584 and 4,760,071, for example, hereby inco ⁇ orated by reference).
  • PROSCAR® which is principally active in prostatic tissue due to its selectivity for human 5- ⁇ reductase isozyme 2
  • combinations of compounds which are specifically active in inhibiting isozyme 1 (found particularly in skin) and compounds which act at both of these isozymes, are useful in combination with compounds identified according to this invention.
  • such a dual inhibitor of 5 ⁇ -reductase 1 and 2 could be used in combination with a 5 ⁇ -reductase 1 inhibitor or with a 5 ⁇ - reductase 2 inhibitor, e.g. finasteride (PROSCAR®), for combination therapy in the treatment of hyperandrogenic conditions, in combination with compounds identified according to this inventionas being selective human alphalC adrenergic receptor antagonists.
  • the dual 5 ⁇ -reductase isozyme inhibitor could also be used in combination with a potassium channel opener, e.g. minoxidil, for the treatment of male pattern baldness, and such combinations in combination with selective human alphalC adrenergic receptor antagonists also form part of the instant invention .
  • the present invention also has the objective of providing suitable topical, oral, systemic and parenteral pharmaceutical formulations for use in the novel methods of treatment of the present invention.
  • compositions containing compounds identified according to this invention as the active ingredient for use in the specific antagonism of human alphalC adrenergic receptors can be administered in a wide variety of therapeutic dosage forms in conventional vehicles for systemic administration.
  • the compounds can be administered in such oral dosage forms as tablets, capsules (each including timed release and sustained release formulations), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by injection.
  • intravenous both bolus and infusion
  • intraperitoneal subcutaneous
  • topical with or without occlusion
  • intramuscular form all using forms well known to those of ordinary skill in the pharmaceutical arts.
  • An effective but non-toxic amount of the compound desired can be employed as an alphalC antagonistic agent.
  • the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult human/per day.
  • the compositions are preferably provided in the form of scored or unscored tablets containing 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, and 50.0 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated.
  • An effective amount of the drug is ordinarily supplied at a dosage level of from about 0.0002 mg./kg to about 50 mg./kg. of body weight per day. The range is more particularly from about 0.001 mg./kg to 7 mg./kg. of body weight per day.
  • the dosages of the alphalC adrenergic receptor and testosterone 5-alpha reductase inhibitors are adjusted when combined to achieve desired effects. As those skilled in the art will appreciate, less 5-alpha reductase inhibitor may be required when the acute symptoms of BPH are alleviated by treatment with alphalC adrenergic receptor inhibitors has been initiated. On the other hand, dosages of these various agents may be independently optimized and combined to achieve a synergistic result wherein the pathology is reduced more than it would be if either agent were used alone.
  • compounds of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three or four times daily.
  • compounds for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art.
  • the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.
  • compounds exhibiting at least 12 fold selectivity for inhibition of the alphalC adrenergic receptor can be combined with a therapeutically effective amount of a 5 ⁇ -reductase 2 inhibitor, such as finasteride, in addition to a 5 ⁇ -reductase 1 inhibitor, such as 4,7 ⁇ -dimethyl-4-aza-5 ⁇ -cholestan-3-one, in a single oral, systemic, or parenteral pharmaceutical dosage formulation.
  • a 5 ⁇ -reductase 2 inhibitor such as finasteride
  • a 5 ⁇ -reductase 1 inhibitor such as 4,7 ⁇ -dimethyl-4-aza-5 ⁇ -cholestan-3-one
  • a combined therapy can be employed wherein the alphalC adrenergic receptor antagonist and the 5 ⁇ -reductase 1 or 2 inhibitor are administered in separate oral, systemic, or parenteral dosage formulations.
  • the compounds of the instant invention and dual inhibitors of 5 ⁇ -reductase 1 and 2 could be formulated for topical administration.
  • niguldipine or 5-methyl urapidil and finasteride can be administered in a single oral or topical dosage formulation, or each active agent can be administered in a separate dosage formulation, e.g., in separate oral dosage formulations, or an oral dosage formulation of finasteride in combination with a topical dosage formulation of a compound exhibiting dual inhibiton of both isozymes of 5 ⁇ -reductase.
  • each active agent can be administered in a separate dosage formulation, e.g., in separate oral dosage formulations, or an oral dosage formulation of finasteride in combination with a topical dosage formulation of a compound exhibiting dual inhibiton of both isozymes of 5 ⁇ -reductase.
  • a potassium channel opener such as minoxidil, cromakalin, pinacidil, a compound selected from the classes of S-triazine, thiane-1 -oxide, benzopyran, and pyridinopyran derivatives or a pharmaceutically acceptable salt thereof
  • compounds of this invention may also be used in combination therapy for the treatment of androgenic alopecia including male pattern baldness.
  • the active agents can be administered in a single topical dosage formulation, or each active agent can be administered in a separate dosage formulation, e.g., in separate topical dosage formulations, or an oral dosage formulation of a compound of formula I in combination with a topical dosage formulation of, e.g., minoxidil. See, e.g., U.S. Patent No.'s 4,596,812, 4,139,619 and WO 92/02225, published 20 February 1992, for dosages and formulations of calcium channel openers.
  • the active agents can be administered concurrently, or they each can be administered at separately staggered times.
  • the dosage regimen utilizing the compounds of the present invention is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound thereof employed.
  • a physician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition.
  • Optimal precision in achieving concentration of drug within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the drug's availability to target sites. This involves a consideration of the distribution, equilibrium, and elimination of a drug.
  • the compounds herein described in detail can form the active ingredient, and are typically administered in admixture with suitable pharmaceutical diluents, excipients or carriers (collectively referred to herein as "carrier” materials) suitably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.
  • carrier suitable pharmaceutical diluents, excipients or carriers
  • the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like.
  • suitable binders, lubricants, disintegrating agents and coloring agents can also be inco ⁇ orated into the mixture.
  • suitable binders include, without limitation, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like.
  • Lubricants used in these dosage forms include, without limitation, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like.
  • Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum and the like.
  • the liquid forms in suitably flavored suspending or dispersing agents such as the synthetic and natural gums, for example, tragacanth, acacia, methyl-cellulose and the like.
  • suspending or dispersing agents such as the synthetic and natural gums, for example, tragacanth, acacia, methyl-cellulose and the like.
  • Other dispersing agents which may be employed include glycerin and the like.
  • glycerin for parenteral administration, sterile suspensions and solutions are desired.
  • Isotomc preparations which generally contain suitable preservatives are employed when intravenous administration is desired.
  • Topical preparations containing the active drug component can be admixed with a variety of carrier materials well known in the art, such as, e.g., alcohols, aloe vera gel, allantoin, glycerine, vitamin A and E oils, mineral oil, PPG2 myristyl propionate, and the like, to form, e.g., alcoholic solutions, topical cleansers, cleansing creams, skin gels, skin lotions, and shampoos in cream or gel formulations. See, e.g., EP 0 285 382.
  • the compounds of the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles.
  • Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.
  • Compounds of the present invention may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled.
  • the compounds of the present invention may also be coupled with soluble polymers as targetable drug carriers.
  • Such polymers can include polyvinyl- pyrrolidone, pyran copolymer, polyhydroxypropylmethacryl- amidephenol, polyhydroxy-ethylaspartamidephenol, or polyethyl- eneoxidepolylysine substituted with palmitoyl residues.
  • the compounds of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydro-pyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.
  • a drug for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydro-pyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.
  • oligonucleotides Based on the amino acid homologies of human ⁇ lA, rat ⁇ lB and bovine ⁇ lC receptors, degenerate oligonucleotides were designed to amplify cDNAs encoding all three receptor subtypes. These oligonucleotides are:
  • WC * (SEQ.ID:3) TTTTCTAGAGAARAANGGNARCCARC 26
  • Oligonucleotides MYC and WL' were used as primers in a reverse transcription PCR amplification of human heart mRNA (Clontech) using the RNA PCR kit from Perkin Elmer Cetus. Briefly, 0.5 ug of mRNA was reverse transcribed in a volume of 20 ul using either random oligonucleotide primers (reaction 1) or oligo dT primer (reaction 2). Reactions 1 and 2 were pooled and served as template for PCR amplification as follows:
  • Prep scale tertiary reaction 3 X 200 ul: 19.5 ul 10X buffer 32 ul 1.25 mM each stock of dATP, dCTP, dGTP, and dTTP 5 ul secondary PCR reaction 4 ul 100 pMoles oligo MYC 4 ul 100 pMoles oligo WC 1 ul 5 units Amplitaq DNA polymerase 134.5 ul water
  • the PCR product was purified by Qiagen spin columns and digested with restriction endonucleases Spel and Xbal. The fragment was then ligated into Spel/Xbal cut pGEM9Zf(-). The ligation mix was used to transform E. coli XL-1 blue. Plasmid DNA was isolated from white transformants and sequenced by the dideoxy chain termination method. The base sequence obtained is shown in Fig. 1, SEQ. ID:4:.
  • a cDNA library prepared from mRNA isolated from human hippocampus (Stratagene) was screened by plaque hybridization using ph ⁇ lX as a probe. Hybridization conditions were as follows:
  • the 3' end of the coding region of human alphalC adrenergic receptor was amplified from human genomic DNA using two oligonucleotides:
  • the PCR product was purified by Qiagen spin columns and digested with restriction endonuclease EcoRI. The fragment was then ligated into EcoRI cut pGEM3Zf(-). The ligation mix was used to transform E. coli XL-1 blue. Plasmid DNA was isolated from white transformants and sequenced by the dideoxy chain termination method. The base sequence is shown in Fig. 4, SEQ. 1D:10:.
  • the complete coding region of human alphal c adrenergic receptor was assembled by ligating the cDNA clone (see Example 2, figure 3, SEQ ID:7:) and 3'CG (see Example 3, figure 4, SEQ 1D:10: ) at their common PvuII site ( 1552-1557 of figure 3, SEQ ID:7: and 59-64 of figure 4, SEQ ID: 10:).
  • the complete nucleotide sequence is shown in figure 5, SEQ ID:11:.
  • the amino acid sequence is shown in figure 6, SEQ. ID: 12:.
  • Figure 7 shows the structure of the cDNA, including the 5'-untranslated sequences.
  • the very 3' twenty seven nucleotides (6 amino acids) shown is the sequence of the PCR primer used to generate the sequence.
  • the complete sequence (SEQ ID:11 :) of the human alphalC adrenergic receptor was subcloned into the eukaryotic expression vector pcDNAI-neo (Invitrogen). The resulting plasmid was transfected into COS -7 cells by electroporation. Cells were harvested after 72 hours and the membranes containing the expressed receptor protein were prepared as described in Schwinn, et al.. J. Biol. Chem.. 265:8183- 8189, 1990.
  • Membranes (5-25 ug, see figure 8) prepared from the COS-7 cells transfected with the vector containing the alphalC receptor gene specifically bound the alpha 1 antagonist [125 I] -HEAT; membranes prepared from the COS -7 cells transfected with the vector alone did not bind the alpha 1 antagonist [125 I] -HEAT (figure 8), proving the expression of the alphalC adrenergic receptor.
  • Reactions were incubated at room temperature for one hour with shaking. Reactions were filtered onto Whatman GF/C glass fiber filters with a Brandel cell harvester. Filters were washed three times with ice cold buffer and bound radioactivity was determined. Non specific binding was determined in the presence of 10 uM prazosin.
  • the complete coding region for the human alphal A adrenergic receptor (Bruno, et al., BBRC. 179:1485-1490, (1991); see figure 10, SEQ. ID:13: and figure 11, SEQ. ID:14: herein) was subcloned into the eukaryotic expression vector pcDNAI-neo (Invitrogen). The resulting plasmid was transfected into COS-7 cells by electroporation. Cells were harvested after 72 hours and the membranes containing the expressed receptor protein were prepared as described in Schwinn, et al.. J. Biol. Chem.. 265:8183-8189, 1990.
  • Oligonucleotides 5XB and A1B were used as primers in a reverse transcription PCR amplification of human heart mRNA (Clontech) using the Invitrogen Copy Kit. Briefly, 1.0 ug of mRNA was reverse transcribed in a volume of 20 ul using oligonuleotide WC as primer.
  • PCR product was directly ligated into pCR vector (Invitrogen) and used to transform E. coli INV ⁇ F' (Invitrogen). Plasmid DNA was isolated from white transformants and sequenced by the dideoxy chain termination method. The base sequence is shown in Fig. 12, SEQ. ID:17:
  • Oligonucleotides EFK and 5B1 were used as primers in a reverse transcription PCR amplification of human aorta mRNA (Clontech) using the Invitrogen Copy Kit. Briefly, 1.0 ug of mRNA was reverse transcribed in a volume of 20 ul using oligo dT as primer.
  • GeneAmp Kit 8 ul 1.25 mM each stock of dATP,dCTP,dGTP, and dTTP 2.0 ul first strand cDNA 1 ul 25 pMoles oligo EFK 1 ul 25 pMoles oligo 5B1 0.25 ul 1.25 units Amplitaq DNA polymersase 33.25 ul water
  • the PCR product was directly ligated into pCR vector (Invitrogen) and used to transform E. coli INVaF (Invitrogen). Plasmid DNA was isolated from white transformants and sequenced by the dideoxy chain termination method. The base sequence is shown in Fig. 13, SEQ. ID:20:.
  • a partial cDNA clone encoding the human alphalB adrenergic receptor was assembled by joining the 5XB sequence (SEQ. ID: 17:) and the EFK sequence (SEQ. ID:20:) at their common BamHI site.
  • S4B SEQ. ID:21: 5' TTT GAA TTC ATG TTC AAG GTG GTG TTC
  • Oligonucleotides S4B and 3'B2 were used as primers in a reverse transcription PCR amplification of rat heart mRNA using the Invitrogen Copy Kit. Briefly, 0.6 ug of mRNA was reverse transcribed in a volume of 20 ul using oligo dT as primer. Primary reaction (50 ul)
  • GeneAmp Kit 8 ul 1.25 mM each stock of dATP,dCTP,dGTP, and dTTP 2.0 ul first strand cDNA 1 ul 25 pMoles oligo EFK 1 ul 25 pMoles oligo 5B1 0.25 ul 1.25 units Amplitaq DNA polymersase 33.25 ul water
  • the PCR product was directly ligated into pCR vector (Invitrogen) and used to transform E. coli INV ⁇ F' (Invitrogen). Plasmid DNA was isolated from white transformants and sequenced by the dideoxy chain termination method. The base sequence is shown in Fig. 14, SEQ. ID:23:.
  • the partial human alphalB adrenergic receptor cDNA was joined to the 3' end of the rat alphalB adrenergic receptor cDNA at their common BssHII restriction endonuclease site.
  • This composite sequence is shown in figure 15, SEQ. ID:24:, and the amino acid sequence is shown in Fig. 16, SEQ. ID:25:
  • the complete coding region for the human/rat alphalB adrenergic receptor was subcloned into the eukaryotic expression vector pcDNAI-neo (Invitrogen). The resulting plasmid was transfected into COS-7 cells by electroporation. Cells were harvested after 72 hours and the membranes containing the expressed receptor protein were prepared as described in Schwinn, et al.. J. Biol. Chem.. 265:8183- 8189, 1990.
  • Binding reactions contained 50 mM Tris-HCl pH. 7.4, 5 mM EDTA, 150 mM NaCl, 100 pM [125 I] -HEAT, and membranes prepared from COS-7 cells transfected with expression plasmids. Reactions are incubated at room temperature for one hour with shaking. Reactions were filtered onto Whatman GF/C glass fiber filters with a Brandel cell harvester. Filters were washed three times with ice cold buffer and bound radioactivity was determined. Non specific binding was determined in the presence of 10 uM prazosin.
  • Membranes prepared from COS-7 cells transfected with the human alpha 1 receptor subtype expression vectors may also be used to identify compounds that selectively bind to the human alphalC adrenergic receptor.
  • 3'CG A 525 bps fragment, specific to complete exon.2 of human alphal c AR, was PCR amplified from human genomic DNA using a sense primer based on the isolated cDNA clone and an antisense primer based on the last six amino acids of bovine alphal c cDNA. This PCR product was subcloned and confirmed by sequencing (see Example 3, SEQ.ID:10:). Genomic Library Screening:
  • DNA was cross-linked with UV cross- linker (Stratagene,La Jolla,CA).
  • IC lambda DNA was amplified by plate lysis method and purified with Qiagen midi-lambda kit (Qiagen,Chatsworth,CA). A 2.6Kb band excised with EcoRI restriction enzyme was identified by Southern analysis using 3'CG probe. This fragment was then subcloned into pGEM3Zf(+) vector.
  • the coding regions differ by a single nucleotide.
  • the genes encode either Cys or Arg at amino acid 347 near the C terminus of the receptor.
  • the nucleotide difference lies within a Pstl restriction enzyme recognition site thus creating a Restriction Fragment Length Polymo ⁇ hism (RFLP).
  • allele 1 LRR
  • allele 2 LCR
  • Allele I is defined by a 2.1 kb Pstl fragment
  • allele 2 yields two bands of 1.6 and 0.5 kb. Since the amino acid difference occurs within the intracellular tail of the receptor we would not expect any pharmacological differences between the expressed receptors.
  • a cosmid library containing FG293 cell line genomic DNA in the double-cos vector sCos-1 was screened as follows: The published human ⁇ i a receptor cDNA clone (Bruno et al., BBRC. 179:1485-1490 (1991), and see Fig. 10, SEQ.ID:13:) was cloned into the vector pcDNAl neo to generate the clone pEX ⁇ la.
  • the filters were incubated with 1 x 10 6 cpm/ml of probe in 5X SSC, 35% Formamide, 0.02% SDS, 0.1 % lauroyl sarcosine, 2% blocking buffer (Bohrenger Mannheim), at 42 °C for 18 hours.
  • the filters were washed with 2 liters of 0.5X SSC, 0.1% SDS, 55 °C and exposed to Kodak XAR-5 film. Twelve primary positives were picked from master plates and re-screened using the ⁇ ia- specific probe.
  • a Cosmid containing ⁇ la receptor exon 1 DNA was subjected to restriction digestion by endonuclease Pst I and subjected to southern blot analysis as above using the ⁇ la -specific probe. Two fragments of 2.3 and 1.6 kb were detected and subcloned into the Pst I site of PGEM 3ZF . The presence of the correct 5' terminal sequences in the 2.3 kb fragment was confirmed by sequencing across the junction between inverted repeat and non-repeat sequences. The 5' end of the ⁇ ia receptor gene was ligated to the cDNA clone at their common Pstl site, see figures 22-24, SEQ.ID:29:, and SEQ.ID:30:.
  • the objective of this assay is to eliminate agents which specifically affect binding of [3H] spiperone to cells expressing human dopamine receptors D2, D3 or D4.
  • Frozen pellets containing specific dopamine receptor subtypes stably expressed ' in clonal cell lines are lysed in 2 ml lysing buffer (lOmM Tris-HCl/5mM Mg, pH 7.4). Pellets obtained after centrifuging these membranes (15' at 24,450 ⁇ m) are resuspended in 50mM Tris-HCl pH 7.4 containing EDTA, MgCl[2], KCl, NaCl, CaCl[2] and ascorbate to give a 1 Mg/mL suspension. The assay is initiated by adding 50-75 ⁇ g membranes in a total volume of 500 ⁇ l containing 0.2 nM [3H]-spiperone.
  • Non-specific binding is defined using 10 ⁇ M apomo ⁇ hine.
  • the assay is terminated after a 2 hour incubation at room temperature by rapid filtration over GF/B filters presoaked in 0.3% PEI, using 50mM Tris-HCl pH 7.4.
  • the objective of this assay is to eliminate agents which specifically affect binding to cloned human 5HTla receptor
  • Mammalian cells expressing cloned human 5HTla receptors are lysed in ice-cold 5 mM Tris-HCl , 2 mM EDTA (pH 7.4) and homogenized with a polytron homogenizer. The homogenate is centrifuged at lOOOXg for 30', and then the supernatant is centrifuged again at 38,OOOXg for 30'.
  • the binding assay contains 0.25 nM [3H]8- OH-DPAT in 50 mM Tris-HCl, 4 mM CaC12 and lmg/ml ascorbate. Non-specific binding is defined using 10 ⁇ M propranolol. The assay is terminated after a 1 hour incubation at room temperature by rapid filtration over GF/Cfilters EXAMPLE 14
  • Taconic Farms Sprague-Dawley male rats, weighing 250- 400 grams are sacrificed by cervical dislocation under anesthesia (methohexital; 50 mg/kg, i.p.). An incision is made into the lower abdomen to remove the ventral lobes of the prostate.
  • Each prostate removed from a mongrel dog is cut into 6-8 pieces longitudinally along the urethra opening and stored in ice-cold oxygenated Krebs solution overnight before use if necessary.
  • Dog urethra proximal to prostate is cut into approximately 5 mm rings, the rings are then cut open for contractile measurement of circular muscles.
  • Human prostate chips from transurethral surgery of benign prostate hype ⁇ lasia are also stored overnight in ice-cold Krebs solution if needed.
  • the tissue is placed in a Petri dish containing oxygenated Krebs solution [NaCl, 118 mM; KCl, 4.7 mM; CaCl2, 2.5 mM; KH2PO4, 1.2 mM; MgS04, 1.2 mM; NaHC ⁇ 3, 2.0 mM; dextrose, 11 mM] warmed to 37°C. Excess lipid material and connective tissue are carefully removed.
  • Tissue segments are attached to glass tissue holders with 4-0 surgical silk and placed in a 5 ml jacketed tissue bath containing Krebs buffer at 37°C, bubbled with 5% C ⁇ 2/95% 02-
  • the tissues are connected to a Statham-Gould force transducer; 1 gram (rat, human) or 1.5 gram (dog) of tension is applied and the tissues are allowed to equilibrate for one hour. Contractions are recorded on a Hewlett-Packard 7700 series strip chart recorder.
  • a cumulative concentration response curve to an agonist is generated; the tissues are washed every 10 minutes for one hour. Vehicle or antagonist is added to the bath and allowed to incubate for one hour, then another cumulative concentration response curve to the agonist is generated.
  • EC50 values are calculated for each group using GraphPad Inplot software.
  • Benign prostatic hype ⁇ lasia causes decreased urine flow rate that may be produced by both passive physical obstruction of the prostatic urethra from increased prostate mass as well as active obstruction due to prostatic contraction.
  • Alpha adrenergic receptor antagonists such as prazosin and terazosin prevent active prostatic contraction, thus improve urine flow rate and provide symptomatic relief in man.
  • these are non-selective alpha- 1 receptor antagonists which also have pronounced vascular effects. Because we have identified the alpha- IC receptor subtype as the predominent subtype in the human prostate, it is now possible to specifically target this receptor to inhibit prostatic contraction without concomitant changes in the vasculature.
  • the following model is used to measure adrenergically mediated changes in intra-urethral pressure and arterial pressure in anesthetized dogs in order to evaluate the efficacy and potency of selective alpha adrenergic receptor antagonists.
  • the goals are to: 1) identify the alpha- 1 receptor subtypes responsible for prostatic/urethral contraction and vascular responses, and 2) use this model to evaluate novel selective alpha adrenergic antagonists. Novel and standard alpha adrenergic antagonists may be evaluated in this manner.
  • the dogs are anesthetized with pentobarbital sodium (35 mg/kg, i.v. plus 4 mg/kg/hr iv infusion).
  • An endotracheal tube is inserted and the animal ventilated with room air using a Harvard instruments positive displacement large animal ventilator.
  • Catheters PE 240 or 260
  • Catheters are placed in the aorta via the femoral artery and vena cava via the femoral veins (2 catheters, one in each vein) for the measurement of arterial pressure and the administration of drugs, respectively.
  • a supra-pubic incision ⁇ l/2 inch lateral to the penis is made to expose the urethers, bladder and urethra.
  • the urethers are ligated and cannulated so that urine flows freely into beakers.
  • the dome of the bladder is retracted to facilitate dissection of the proximal and distal urethra.
  • Umbilical tape is passed beneath the urethra at the bladder neck and another piece of umbilical tape is placed under the distal urethra approximately 1-2 cm distal to the prostate.
  • the bladder is incised and a Millar micro-tip pressure transducer is advanced into the urethra.
  • the bladder incision is sutured with 2-0 or 3-0 silk (purse-string suture) to hold the transducer.
  • Phenylephrine an alpha- 1 adrenergic agonist
  • Phenylephrine an alpha- 1 adrenergic agonist
  • phenylephrine dose-response curves are generated in each animal (one control, three or four doses of antagonist or vehicle).
  • the relative antagonist potency on phenylephrine induced changes in arterial and intra-urethral pressure are determined by Schild analysis.
  • the family of averaged curves are fit simultaneously (using ALLFIT software package) with a four paramenter logistic equation constraining the slope, minimum response, and maximum response to be constant among curves.
  • the dose ratios for the antagonist doses (rightward shift in the dose-response curves from control) are calculated as the ratio of the ED50's for the respective curves.
  • the Kb dose of antagonist causing a 2-fold rightward shift of the phenylephrine dose-response curve
  • the relative selectivity is calculated as the ratio of arterial pressure and intra-urethral pressure Kb's. Effects of the alpha- 1 antagonists on baseline arterial pressure are also monitored.
  • Comparison of the relative antagonist potency on changes in arterial pressure and intra-urethral pressure provide insight as to whether the alpha receptor subtype responsible for increasing intra-urethral pressure is also present in the systemic vasculature. According to this method, one is able to confirm the selectivity of alphalC adrenergic receptor antagonists that prevent the increase in intra-urethral pressure to phenylephrine without any activity at the vasculature.
  • the dogs are killed via an overdose of intravenously administered pentobarbital or saturated KCl.
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO:l: TTTTCTAGAT TRTTNARRTA NCCNAGCC 28
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • GAATTCCCTC CTAGAAGCTG GAGAGAGCAG GAGCCTTCGG TGGGGCAGCT CAAAATGTAG 60
  • AACATTTCCA AGGCCATTCT GCTCGGGGTG ATCTTGGGGG GCCTCATTCT TTTCGGGGTG 720 CTGGGTAACA TCCTAGTGAT CCTCTCCGTA GCCTGTCACC CACTACTACA TCGTCAACCT GGCGGTGGCC GACCTCCTGC TTCTCCGCCA TCTTCGAGGT CCTAGGCTAC TGGGCCTTCG TGGGCGGCAG TGGATGTGCT GTGCTGCACC GCGTCCATCA ATCGACCGCT ACATCGGCGT GAGCTACCCG CTGCGCTACC AGGGGTCTCA TGGCTCTGCT CTGCGTCTGG GCACTCTCCC CTCTTCGGCT GGAGGCAGCC GGCCCGAG GACGAGACCA CCGGGCTACG TGCTCTTCTC GGCTCTGGGC TCCTTCTACC GTCATGTACT GCCGCGTCTA CGTGGTGGCC AAGAGGGAGA CTCAAGACCG ACAAGTCGGA CTCGGAGCAA GTGACGCTCC CC
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI -SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • AACATTTCCA AGGCCATTCT GCTCGGGGTG ATCTTGGGGG GCCTCATTCT TTTCGGGGTG 720
  • TTCTCCGCCA TCTTCGAGGT CCTAGGCTAC TGGGCCTTCG GCAGGGTCTT CTGCAACATC 900
  • TCCATGCCCC GTGGATCTGC CAGGATTACA GTGTCCAAAG ACCAATCCTC CTGTACCACA 1860 GCCCGGGTGA GAAGTAAAAG CTTTTTGCAG GTCTGCTGCT GTGTAGGGCC CTCAACCCCC 1920
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE N-terminal
  • CAAGGTCATC TTCTGGCTCG GCTACTTCAA CAGCTGCGTG AACCCGCTCA TCTACCCCTG 1140
  • CGCCAGCCGT CGAAGCCACC CAGCGCCTTC CGCGAGTGGA GGCTGCTGGG GCCGTTCCGG 1440
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE CDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI -SENSE NO
  • AGAACTTTCA CGAGGACACC CTTAGCAGTA CCAAGGCCAA GGGCCACAAC CCCAGGAGTT 840 CCATAGCTGT CAAACTTTTT AAGTTCTCCA GGGAAAAGAA AGCAGCTAAG ACGTTGGGCA 900
  • CCTTGTTCTC CACCCTGAAG CCCCCCGACG CCGTGTTCAA GGTGGTGTTC TGGCTGGGCT 1020
  • GAATTCCCTC CTAGAAGCTG GAGAGAGCAG GAGCCTTCGG TGGGGCAGCT CAAAATGTAG 60
  • AACATTTCCA AGGCCATTCT GCTCGGGGTG ATCTTGGGGG GCCTCATTCT TTTCGGGGTG 720
  • AATTCCCTCC TAGAAGCTGG AGAGAGCAGG AGCCTTCGGT GGGGCAGCTC AAAATGTAGG 60
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • CTCCCTGCCG GCCGCTCGTT CTGTGCCCCG GCCCGGCCAC CGACGGCCGG CGTTGAGATG 60
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO:31: GAATCCCGAC CTGGAC 16
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO

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Abstract

Le récepteur adrénergique humain de sous-type alphaC est cloné et utilisé dans une méthode de criblage in vitro de composés se fixant spécifiquement au récepteur alpha1C-adrénergique humain, y compris de composés efficaces pour l'atténutation des symptômes de l'hypertrophie prostatique. L'invention concerne la méthode, le récepteur humain cloné utilisé dans cette dernière, un récepteur alpha1C-adrénergique humain isolé, exempt de toute autre protéine humaine, et des composés identifiés à l'aide de ce nouveau récepteur cloné, qui fixent sélectivement le récepteur alpha1C-adrénergique humain.
PCT/US1994/002609 1993-03-15 1994-03-10 Recepteur alpha1c-adrenergique humain clone WO1994021660A1 (fr)

Priority Applications (3)

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JP6521115A JPH08508163A (ja) 1993-03-15 1994-03-10 クローン化ヒトアドレナリンα１Ｃ受容体
AU64453/94A AU685789B2 (en) 1993-03-15 1994-03-10 Cloned human alphaic adrenergic receptor
EP94912209A EP0689547A4 (fr) 1993-03-15 1994-03-10 Recepteur alpha1c-adrenergique humain clone

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JP (1) JPH08508163A (fr)
AU (1) AU685789B2 (fr)
CA (1) CA2158345A1 (fr)
WO (1) WO1994021660A1 (fr)

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US5661163A (en) * 1995-06-07 1997-08-26 Merck & Co., Inc. Alpha-1a adrenergic receptor antagonists
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Life Sciences, Volume 37, Number 25, issued 1985, COTECCHIA et al., "Phorbol Ester Effects on Alpha1-Adrenoceptor Binding and Phosphatidylinositol Metabolism in Cultured Vascular Smooth Muscle Cells", pages 2389-2398, see pages 2391-2395. *
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Cited By (15)

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US5578611A (en) * 1992-11-13 1996-11-26 Synaptic Pharmaceutical Corporation Use of α-1C specific compounds to treat benign prostatic hyperplasia
US6602888B2 (en) 1992-11-13 2003-08-05 Synaptic Pharmaceutical Corporation Use of α1C specific compounds to treat benign prostatic hyperplasia
US6015819A (en) * 1992-11-13 2000-01-18 Synaptic Pharmaceutical Corporation Use of alpha-1C specific compounds to treat benign prostatic hyperplasia
US5780485A (en) * 1992-11-13 1998-07-14 Synaptic Pharmaceutical Corporation Use of α1c specific compounds to treat benign prostatic hyperplasia
US5990128A (en) * 1994-04-13 1999-11-23 Synaptic Pharmaceutical Corporation α1C specific compounds to treat benign prostatic hyperplasia
US5952351A (en) * 1995-02-23 1999-09-14 Merck & Co., Inc. Alpha 1a adrenergic receptor antagonists
US6096763A (en) * 1995-02-23 2000-08-01 Merck & Co., Inc. α1a adrenergic receptor antagonists
US5668148A (en) * 1995-04-20 1997-09-16 Merck & Co., Inc. Alpha1a adrenergic receptor antagonists
US5977115A (en) * 1995-06-07 1999-11-02 Merck & Co., Inc. Alpha 1a adrenergic receptor antagonists
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JPH08508163A (ja) 1996-09-03
EP0689547A4 (fr) 1998-10-28
CA2158345A1 (fr) 1994-09-29
AU685789B2 (en) 1998-01-29
AU6445394A (en) 1994-10-11
EP0689547A1 (fr) 1996-01-03

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